The objective of the proposed research is to understand how information concerning environmental phosphate levels is transmitted through the phosphate signal transduction pathway to control gene expression in Escherichia coli. At the heart of this pathway are the response regulator PhoB and the histidine kinase PhoR. A great deal of work has already been accomplished elucidating the mechanisms by which these two proteins function. The focus of this proposal is to understand how two auxiliary proteins, PstSCAB2 and PhoU, are involved in the sensing of phosphate and the control of this signaling pathway. Given the history of research into phosphate regulation, it is somewhat surprising that fundamental questions still exist as to how cells sense phosphate and how that information is transmitted within a cell. PstSCAB2 is a phosphate transporter that also functions as the sensor for the system. PhoU controls the activity of PstSCAB2 and is required for the transmission of the signal from PstSCAB2 to PhoR. Our first hypothesis is that the PstSCAB2 transporter transduces information about environmental phosphate levels through conformational changes that are inherent to the transport process. Our second hypothesis is that PhoU transmits this information by specifically interacting with a particular PstSCAB2 conformation and mediates protein/protein interactions with PhoR. We plan on addressing these hypotheses through both genetic and biochemical approaches. Mutant versions of the transporter will be isolated that "lock" it into various conformations. We will use these versions of the transporter to trap complexes with other proteins using co-elution and co-immunoprecipitation experiments and as well as bacterial two-hybrid analysis. These approaches should prove to be complementary to one another and will provide the greatest opportunity to observe protein/protein interactions within the proposed signaling complex. In the analysis of the role of PhoU, constitutive signaling mutants will be isolated in the absence of the transporter and studied for interactions with PhoR. In addition, highly conserved amino acid residues of PhoU, which must be important to its function, will be mutated and studied for function and cellular localization. PhoU-GFP fusions will also be constructed and used to study localization. The proposed work is important because these signaling proteins are essential for bacteria to survive changing environments - including the human immune system. This feature combined with their absence in higher eukaryotes, makes this signaling pathway a targets for the development of new antimicrobial drugs. An increased understanding of these signal transduction proteins may assist in the rational design of drugs to combat pathogens. PUBLIC HEALTH RELEVANCE: This project, which focuses on determining how bacterial cells sense phosphate to control various survival genes, is relevant to public health because it provides foundational basic research into a fundamental process of bacteria. The phosphate sensory response pathway is essential to many pathogens in their ability to cause disease. Because of this research, strategies and drugs may be developed that will limit a pathogens survival, thereby helping to combat various bacterial infections.